Among novel therapies being explored for malignant glioma, oncolytic viruses possess relative tumor selectivity and are being tested in clinical trials. In prior years of funding, we have constructed herpes simplex viruses genetically engineered to replicate in tumor cells. Published phase I clinical trials for brain tumors have reported the safety of herpes viral vectors but offered little data concerning efficacy in humans. We have found that herpes viral replication can be enhanced by the drug, cyclophosphamide. In published and pilot experiments, the effect of cyclophosphamide (CPA) is rapid and does not require the presence of pre-existing immunity to the oncolytic virus. CPA leads to increased viral liters in multiple types of glial tumors (human, mice or rat) and results in increased survival. The effect appears to also occur with an oncolytic adenovirus. We hypothesize that normal host responses impede the process of oncolytic viral infection. In pilot data, macrophages appear to be a major determinant of such host responses. We have also dentified molecular markers (including IFNy, iNOS, VEGFR2, APN and versican) of the host (and possibly macrophage-mediated) response to oncolytic viral infection and have preliminary data that CPA acts as a sensitizer of viral oncolysis by limiting such responses. The CPA effect is relatively pleiotropic and there is a need to identify specific pathways that underlie the host processes that impede viral oncolysis and pharmacologic means to limit such responses. We thus propose the following aims: AIM 1-Validate changes in the molecular markers that are associated with viral oncolysis;AIM2- Determine the cellular infiltrates in tumor that are associated with viral oncolysis;AIM 3-Determine if CD68+ cells (macrophages/microglia) and their gene products limit viral oncolysis: AIM 4-Determine if CD56+ (NK cells) and their gene product, IFNY, limit viral oncolysis. We will employ quantitative RT-PCR, FACS, molecular imaging (Project 3, immunohistochemistry (Pathology Core) and genetic and pharmacologic analyses to prove or refute the stated hypotheses, using GLP- and GMP-grade oncolyic HSV (Vector Core B). If successful, this project will begin to elucidate at a molecular level how the host responds to a viral infection of its brain tumor and how this can be modulated to increase anticancer effects. This should be of significance for current and future clinical trials of cancer that employ this modality.